title: Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.
abstract: Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.
authors.affiliation: Department of Dermatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: M J
authors.initials: MJ
authors.lastname: Fazio
authors.forename: V M
authors.initials: VM
authors.lastname: Kähäri
authors.forename: M M
authors.initials: MM
authors.lastname: Bashir
authors.forename: B
authors.initials: B
authors.lastname: Saitta
authors.forename: J
authors.initials: J
authors.lastname: Rosenbloom
authors.forename: J
authors.initials: J
authors.lastname: Uitto
mesh_terms: D001483:Base Sequence; D004549:Elastin / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic
chemical_list: D004549:Elastin